[eng] A new design of permeation module based on 3D printing was developed to monitor the interaction of exogenic compounds with cell membrane transporters in real-time. The fluorescent marker Rhodamine 123 (Rho123) was applied as a substrate to study the activity of the P-glycoprotein membrane transporter using the MDCKII-MDR1 genetically modified cell line. In addition, the inhibitory effect of verapamil (Ver), a prototype P-glycoprotein inhibitor, was examined in the module, demonstrating an enhanced Rho123 transfer and accumulation into cells as well as the applicability of the module for P-glycoprotein inhibitor testing. Inhibition was demonstrated for different ratios of Rho123 and Ver, and their competition in terms of interaction with the P-glycoprotein transporter was monitored in real-time. Employing the 3D-printed module, permeation testing was shortened from 8 h in the conventional module to 2 h and evaluation based on kinetic profiles in every 10 min was possible in both donor and acceptor compartments. We also show that monitoring Rho123 levels in both compartments enables calculate the amount of Rho123 accumulated inside cells without the need of cell lysis.